Synthesis of novel pyrazolone candidates with studying some biological activities and in-silico studies

Pyranopyrazole derivatives have a vital role in the class of organic compounds because of their broad spectrum of biological and pharmacological importance. Our current goal is the [3 + 3] cycloaddition of benzoyl isothiocyanate and pyrazolone 1 to undergo oxidation cyclization, producing pyrazoloxadiazine 3. The diol 5 was obtained as a condensation of two equivalents of 1 with thiophene-2-carboxaldehyde in acetic acid above the sodium acetate mixture. When the condensation was carried out in piperidine under fusion, unsaturated ketone 4 was obtained. The pyrazolo pyran derivative 11 resulted from the [3 + 3] cycloaddition of 1 and cinnamic acid, while the Pyrone derivative was prepared by acylation of 12 with two equivalents of acetic anhydride. Phthalic anhydride undergoes arylation using zinc chloride as a catalyst. The cyclic keto acid 23 was synthesized by the action of succinic anhydride on 12 in the acetic medium, while the latter reacted with cinnamic acid, leading to pyrazole derivative 24. All of these reactions were through the Michael reaction mechanism. All the tested compounds showed good antimicrobial activity against pathogenic microorganisms; newly synthesized compounds were also screened for their antioxidant activity. Rational studies were carried out by the ABTs method to allow a broader choice of activities. In addition, similar off-compounds were conducted. Molecular docking studies with the CB-Dock server and MD simulations were created with the default settings of the Solution Builder on the CHARMM-GUI server at 150 nm. A good correlation was obtained between the experimental results and the theoretical bioavailability predictions using POM theory.

www.nature.com/scientificreports/In this context, the pyrazole derivatives skeleton is a fertile source of biologically important molecules possessing a broad spectrum of biological and pharmacological activities such as anti-inflammatory and transformations of condensed heterocyclic derivatives are of theoretical interest for developing new synthetic methods and studying the relationships between chemical structure and reactivity of organic compounds 1 .In addition, pyrazole-based heterocyclic ligands have multiple biological applications.For example, many compounds prepared have high efficiencies as antibacterial or antifungal candidates due to their nitrogen electron and proton acceptor abilities 2 .Furthermore, the presence of the pyrazole nucleus in different structures leads to diversified applications in different areas such as technology, medicine, and agriculture.Furthermore, pyrazole derivatives have arrived an extensive view of researchers through the past few decades due to their highly reactive effects as anti-inflammatory 3 , antiglaucoma 4 , antiviral 5 , antimicrobial 6 , antidiabetic activities 7 .In addition, pyrazole prodrugs have been recorded to maintain significant anticancer activity [8][9][10][11][12] .Pyrazole nucleus is a rare structural stage attractive for combinatorial and medicinal chemistry.In addition, it encloses the most recent reports on a structural variation on pyrazole explaining vital structural activity relationship 13 .Whereas the late-stage explanation of a pyrazole ring through some cycloadditions of previously substituted components is the basis for most substituted pyrazoles 14,15 , direct functionalization has not been examined satisfyingly to date.As analysis on it seems rare, we have been interested in and examined the direct functionalization of pyrazoles through coupling reactions of halogenated analogs taken from commercially available pyrazole [16][17][18][19] and prompted by the observed biological activities of the derivatives mentioned above and in Continuation of our ongoing studies on novel biologically active molecules [20][21][22][23] .Nowadays, pyrazole systems, as biomolecules, have attracted more attention due to their interesting pharmacological properties.This heterocycle can be traced in a few well-established drugs belonging to different categories with diverse therapeutic activities.Herein, compound 1 was used as a key intermediate for the synthesis of oxazine, pyrazolotriazinone, and pyrazinopyrimidine derivatives in high yield and purity, to investigate their antimicrobial activity and antioxidant activities (Fig. 1).
With limited facilities to investigate more experimental acceptor abilities, molecular docking became crucial for studying the binding modes and affinities between the prepared compounds and selected biological targets using the lock and key concept 2 .The toxicity predictions and the Lipinski rule of five agreements were determined by POM analysis, one of the well-known approaches to accessing synthetic drugs' pharmacokinetic properties.It helps identify and indicate the type of pharmacophore site that affects biological activity, wherever there may have been changes in the chemical substitution 24,12 .At the same time, MD studies verified the Molecular docking and toxicity prediction results.The impact of molecular dynamics (MD) simulations in molecular biology and drug discovery has expanded dramatically in recent years.These simulations capture the behavior of proteins and other biomolecules in full atomic detail and at excellent temporal resolution 25 .

Rational of the work
We effectively synthesized a variety of beneficial antioxidants using a molecular hybridization process by choosing to reduce functional groups [Fused Pyrazoles] that are directly related to the reducing ascorbic acid ring Fig. 2A and examined them by the ABTs method.Molecular hybridization was proposed to generate an additive or synergistic impact.In addition, it gives a broader choice of activities 26,27 .Figure 2B represents the similar compounds evaluated and induced by the LabMol server.Chemically similar compounds often bind biologically diverse protein targets, and protein structures do not always recognize identical ligands.Pharmacological and off-target relationships among proteins and a ligand set similarity help to improve the machine learning confidence by interpolating the output prediction equalized by the compound similarity criteria.This pipeline help to improve the predictions of off-target drug effects, reducing the false negative error.Chemical similarity is one of the essential concepts in cheminformatics.One commonly used algorithm to calculate these similarity measures is the 2D Tanimoto algorithm employed here.The resulting Tanimoto coefficient is fingerprint-based, encoding each molecule to a fingerprint "bit" position (MACCS), with each bit recording the presence ("1") or absence ("0") of a fragment of the molecule 28,29 .

Chemistry
Cyclic keto methylene units constitute a precursor for the hetero cyclization system.The present article involved the conversation of a pyrazole-bearing keto methylene system to azolo and amino pyrazole of potential biological activities 30,31 .Pyrazole derivative 1 added cyclic nucleophilic nitrogen to activated hetero carbon; then intramolecular cyclodehydration formed an oxadiazine ring affording pyrazolo oxadiazine 3.  www.nature.com/scientificreports/isomers was more deshielded by the 4-oxo group of the thiazole moiety as compared with the E-counterparts and appeared at the lower field (δ ≈ 8.00-8.20 ppm) relative to the E-isomer (δ ≈ 7.50-7.80ppm) (Fig. 3).The IR spectrum of compound 5 revealed OH, NH peak at 3350, 3550 cm −1 , and C=N was observed at 1598 cm −1 .The Contrary to the above result, when thiophene-2-carboxaldehyde was allowed to interact with pyrazolone 1 in a primary medium resulted in a condensation product 4 affording α, β unsaturated system (Fig. 3).Compound 4 showed a conjugated carbonyl group frequency at 1649 cm −1 and a stretching frequency at 1598 cm −1 for an exocyclic double bond.The 1 HNMR spectra showed Olefinic proton at 4.98 ppm in addition to methyl proton at 2.49 ppm and showed two doublet signals at 7.28 ppm and 7.94 ppm due to thiophenyl -C 4 H and thiophenyl  The process may proceed via forming aza Michael followed by amination, cyclo dehydration, and subsequent enolization (Fig. 4). 1 H NMR spectrum of condensed system 7 revealed exchangeable signal at 13.10 ppm for OH proton in addition to Olefinic proton that located at 6.91 ppm, methylene proton at 2.49 ppm and methyl proton at 3.36 ppm while IR spectrum showed broad band 3350-3550 for (OH) cm −1 in addition to a stretching frequency at 1598 cm −1 for exocyclic double bond.The cycloaddition of cinnamic acid and pyrazolone 1 was achieved by H 2 SO 4 as catalyst [activated the carbonyl function in addition to the protonation of pyrazolo nitrogen], resulting in pyran cyclization furnished pyrano pyrazole derivative 11 starting with Michael adduct through 1, 11 additions followed by elimination of H 2 O.The pyran 11 showed C=N at 1683 cm −1 , and the 1 H NMR contained a deshielded signal at 12.35 ppm for OH proton, methyl proton at 2.49, and olefinic proton at 6.53 ppm (Fig. 4).
N-phenyl pyrazolone 12 was reacted with thiophene-3-carboxaldehyde, forming diol derivative 13 (Fig. 5).The OH frequency proved the reaction product 13 at 3460 cm −1 and C=N at 1677 cm −1 . 1 H NMR revealed a downfield signal at 10.75 ppm for OH and methyl proton at 2.39 ppm.Up on condensation of active methylene of ethyl acetoacetate with pyrazole derivative 13 furnished polycyclic compound 19, the reaction involves the formation of ketoester 17 ketonic hydrolysis followed by ester hydration and subsequent aromatization (Fig. 5) the chemical structure of the product was potentiated with the presence of carboxylic carbonyl at 1677 cm −1 and OH at 3431 cm −1 , The a presence of exchangeable deshielded signal at 10.78 ppm for carboxylic proton in addition to methyl group was located at 2.41ppm The carbonyl carbon was detected at 164.1 Diol 13 was reacted with thiosemicarbazide, producing thioimide product 14 (Fig. 6) via condensing two nitrogen and OH group terminals.Thioimide 14 leads to a stretching frequency at 3426 and 1270 cm −1 for NH and C=S, respectively.The thioimide proton signal was downfield at 10.80 ppm, and methyl groups were at 2.43 and 2.49 ppm.When semicarbazide was allowed to be condensed with diol 13, the urea derivative15 was formed in Fig. 6.The IR spectrum of compound 15 displayed as bands at 3430, 1679, and 1636 cm −1 for NH, C=O, and C=N groups 1 H NMR revealed NH proton at 10.80 and 9.84 ppm, in addition to pyridine proton that located at 3.35 ppm While in 13 C The carbonyl carbon signal was showed at 162.78 ppm.Up on heating compound, 13 with ammonium acetate and acetic acid mixture afforded amination followed by pyridine cyclization resulting in di pyrazolo pyridine derivative 16 (Fig. 6) pyridine 16 revealed peaks at 3454 and 1677 cm −1 for NH and C=N.Moreover, condensed pyridine 16 showed in 1 HNMR two doublet signals at 7.28 ppm and 7.94 ppm.This may be due to thiophenyl -C 4 H and thiophenyl -C 3 H and another at 8.11ppm due to thiophenyl -C 5 H and the exchangeable downfield signal at 10.78 ppm for NH and the 3rd proton at 2.46 ppm.Acylation of compound 12 was achieved by reaction with acetic anhydride mixture producing the target 21 displayed carbonyl frequencies at 1677 cm −1 , 1 H NMR displayed aromatic multiplet in addition to methyl ester at 2.30 ppm while pyrazole proton was absolved at 2.49 ppm.Pyrone derivative 21 formed ester 20, followed by acid-catalyzed cyclodehydration (Fig. 7).The phthalic anhydride and N-phenyl pyrazolone 12 with Lewis's acid resulted in acid derivative 22. Compound 22 showed 3460, 1756, and 1677 cm −1 peaks for OH and carbonyl function, respectively.The exchangeable signal at 10.77 was attributed to the carbonylic proton and the pyrazole proton above and below the plane of the ring that decimated at 3.33 and 2.43 ppm.Succinic anhydrides undergo acylation reaction with compound 12 under an acidic medium to furnish keto acid 23; the keto acid compound

Antioxidant evaluation
The antioxidant activities of the synthesized compounds were determined and listed in Table 1 and Fig. 3.The results revealed that all compounds were found to be potent.Moreover, the results showed that nearly compound 5 was found to have the most potent activity levels.Compounds 13, 14, 16, 22, 23, and 24 also had moderate activity.While compound 14 was found to be the lowest potent level.The following points were noticed.Comparing compound 5 and the other compounds showed that compound 5 indicated that the presence of the 2 OH group was more effective than the other compounds.On the other hand, when C=S in compound 14, antioxidant activity decreases.While compounds 24, 22, and 23 were more active than compound 14 due to the COOH group's presence (Fig. 9).
Another method was used to determine the Inhibition by recognition time to achieve maximum Inhibition, which means antimicrobial studies were carried out by measuring the growth inhibition time and taking the     www.nature.com/scientificreports/mean of Inhibition by time method 32,33 .This method indicates the mean time of Inhibition.This test was carried out on the highly active compound observed in both the MIC test and the computational studies.Table 3 and Fig. 11 showed in-vitro experiments for the mean antimicrobial effects on G-positive bacteria S. aureus, revealing.The mean antimicrobial effects were calculated.The same procedure was repeated on G-Negative bacteria E. coli, and the results are indexed in Table 4 and Fig. 12.At the same time, in-vitro experiments for the mean of antifungal effects on C. Albicans and the results are indexed in Table 5 and Fig. 13.Bacteria have developed resistance strains against currently available antimicrobial agents and synthesize novel antimicrobials with less toxicity and more potent effects in less time.The minimal inhibitory concentration for some of the   www.nature.com/scientificreports/newly synthesized compounds showed highly significant activity.Among the screened compounds, 5, 13, and 16 exhibited intense antimicrobial activity 34 .

Minimal inhibitory concentration (MIC)
In general, our synthesized derivatives showed activity against the tested Gram-positive bacteria and the Gramnegative bacteria.The data obtained from the previous two methods showed that compounds 13 and 16 were the most active.Compounds 13 and 16 were investigated again using the MIC test against two-gram positive bacteria (Staphylococcus aureus, Bacillus subtilis) and Gram-negative bacteria (Escherichia coli, Pseudomonas aeruginosa) (Table 6).www.nature.com/scientificreports/ the changes of compounds at the target protein active site.As shown in Fig. 15A, the CCP & 13 and CCP & 16 complexes remained stable in the active pocket after the first 20 ns of pre-MD simulation with mean values below 0.4 nm and 0.27 ± 0.03 and 0.32 ± 0.05 nm in both compounds, respectively.In addition, H bond number measurement was also measured over time, another way of analyzing protein-ligand interactions.After the first 20 ns of pre-MD simulation, as shown in Fig. 15B, there was a continuous H bond formation ranging from one to two between compounds 13 and 16 with CCP Binding mode analysis was performed, as shown in Fig. 15C,D, to analyze protein-ligand interactions at the end of 150 ns and compare them with the docking pose.When the docking pose and the binding poses at the end of the MD simulation were compared, it was concluded that both compounds formed strong and stable interactions at the CCP active site.
In the second MD trajectory analysis section, the radius of gyration (Rg) analysis was performed to explain the compactness of protein-ligand complexes.As shown in Fig. 16A, both CCP & 13 and CCP & 16 protein-ligand complexes were extremely stable around 1.90 nm for 150 ns.Root mean square fluctuation (RMSF) measurements were made to examine the flexibility of the protein structure.As shown in Fig. 16B, the RMSF value was below 0.2 nm except for the protein C and N terminal residues.Active site residues around His175, Leu171, Met172, and Leu177 fluctuated below 0.1 nm for both protein-ligand complexes.MD animation videos in Supporting Information Videos S1 and S2 were created at 150 ns.

POM study
The POM theory is among the most effective platforms because it can fundamentally and efficiently process all organic and organometallic compounds.Tanks of this theory, the design, optimization, and identification of pharmacophore sites (anticancer/antibacterial/antifungal/antiparasitic/ antiviral) depend on every site's physical and chemical properties and Mulliken charges analysis of heteroatoms 35 .
The cLogP and TPSA (Molecular Polar Surface Area) were calculated by using the Osiris program (Tables 7,  8).These two parameters have been considered among the essential descriptors to determine drug absorption, involving each drug molecule's bioavailability, intestinal absorption, and lipophilicity 36 .
The cLogP and TPSA calculations were done for all prepared compounds by POM programs.The predicted cLogP values are less than 5 for all tested molecules except compound 14 has exhibited a clogP of more than 9. Therefore, the values for all studied compounds are less than 5, except for 14.That represents the higher limit for the drug, which can pass into bio-membranes and react with all pockets conforming to the five rule (Lipinski's rule).Based on cLogP values, the prepared compounds are expected to give adequate water solubility and bioavailability (Tables 7, 8).In addition, most of the tested compounds do not exhibit any hazards or toxicities.Compounds 13 and 14 have shown tumorigenic, mutagenic, and reproductive effects of risks of toxicities.However, all tested compounds have generally illustrated great safety and excellent agreement with experimental results and are in an acceptable range compared to the five rules (Tables 8, 9).(5, 16, 13, 22 Antibacterial) We  www.nature.com/scientificreports/have remarked on the presence of many pharmacophore sites (Fig. 17).Most are (-NH + ; -O δ− ) antibacterial sites.Next are (-O δ− ; -O δ− ) antifungal and pharmacophore sites (Fig. 18) 27,37 .

Materials and methods
All chemicals were purchased from Sigma-Aldrich (Taufkirchen, Germany), and all solvents were purchased from El-Nasr Pharmaceutical Chemicals Company (analytical reagent grade, Egypt).All chemicals were used as supplied without further purification.The melting points were measured by a digital Electrothermal IA 9100 Series apparatus Cole-Parmer, Beacon Road, Stone, Staffordshire, ST15 OSA, UK) and were uncorrected.The spectral analysis was performed at KAUST and Mansura Univesity laboratories C, H, and N analyses on a PerkinElmer CHN 2400.In addition, 1 H and 13 C NMR spectra were recorded at KAUST on a Bruker 800 MHz NMR Spectrometer using tetramethylsilane (TMS) as the internal standard, chemical shifts were expressed in δ (ppm), and DMSO-d 6 was used as the solvent.

Figure 2 .
Figure 2. (A) Rationale of the synthesized fused pyrazoles derivatives using molecular hybridization.(B) The similar off compounds of the new series.

Figure 10 .
Figure 10.In-vitro antibacterial and antifungal activity screening assay.

Figure 12 .
Figure 12.The mean percent % of bactericidal in time by *hr.

Figure 13 .
Figure 13.The mean percent % of bactericidal in time by *hr for compound 13.

Figure 16 .
Figure 16.(A) Radius of gyration (Rg) plot describing the compactness of the protein-ligand complexes CCP & 13 and CCP & 16, and (B) root mean square fluctuation (RMSF) plot showing the flexibility and mobility of the protein per residue for 150 ns.

Figure 18 .
Figure 18.Identification of antibacterial and antifungal pharmacophore sites.

Table 1 .
Antioxidant assay for the tested new compounds.

Table 2 .
In-vitro antibacterial and antifungal screening of the newly synthesized compounds. CompoundE.

Table 3 .
The mean percent of bactericidal in time by *h.Molecular dockingMolecular docking studies were used to predict in silico how newly synthesized compounds interact with target proteins.In this context, active compounds 13 and 16 interactions with the cytochrome c peroxidase (CCP) enzyme selected as the target protein were analyzed.Compounds 13 and 16, given in Table7, produced − 9.1 kcal/mol and − 9.6 kcal/mol interaction energies, while CCP's cocrystal ligand ascorbic acid gave − 5.9 kcal/mol interaction energies.As shown in Table7, atomic-level interactions, interaction distances, and types of CCP & 13 and CCP & 16 complexes were analyzed.Both compounds exhibited more potent interactions with the target Compound/time 1 h 3 h 5 h 7 h The mean (%)Vol:.(1234567890)Scientific Reports | (2023) 13:19170 | https://doi.org/10.1038/s41598-023-43575-zwww.nature.com/scientificreports/Computational studies protein CCP than the standard ascorbic acid.As given in Fig. 14, compound 13 conferred an H bond with His75, while compounds 13 and 16 formed an unfavorable positive-positive interaction with Arg48.Molecular dynamics simulations Molecular dynamics (MD) simulations were performed to investigate the stability of CCP & 13 and CCP & 16 complexes obtained with AutoDock Vina.Root means square deviation (RMSD) analysis and H bond analysis between protein and ligand, two important trajectory analysis parameters of MD simulation, carried out for 150 ns duration, were performed.Fitting ligands performed RMSD analysis to protein backbone atoms to analyze

Table 4 .
The mean percent % of bactericidal in time by *h.

Table 5 .
The mean percent of bactericidal in time by *h.

Table 6 .
Antimicrobial activity of compounds.

Table 7 .
Protein-ligand interaction energies and details of the compounds 13 and 16 with target protein cytochrome c peroxidase (CCP).

Table 8 .
Risk of toxicity and drug score predictions.
Figure 17.Atomic charges of tested compounds.